Identification and cloning of highly epitopic regions of Clostridium novyi alpha toxin

Abstract

Introduction Clostridium novyi causes gangrene in humans and animals. Among the various toxins produced by this pathogen (Hatheway, 1990), alpha toxin has a molecular weight of approximately 200-250 kDa and enzymatic activity on Rho receptors in mammalian cells, which cause vascular permeability and cell rounding in culture (Bette et al., 1991). Homology studies have shown that alpha toxin belongs to the large clostridial cytotoxin family (Hofmann et al., 1995). This family comprises glycosyltransferase by modifying small GTP-binding proteins, such as Rho and Ras, which inhibit signal pathways, thereby causing cytoskeletal disintegration (Oksche et al., 1992), which in turn causes cell death. Alpha toxin has a tripartite structure in which the enzymatic activity is related to a 551-amino acid on the N-terminal, known as the catalytic domain (Busch et al., 2000). The C-terminal of the toxin is the receptor-binding domain, and the hydrophobic domain in the central region is required for the translocation of the toxin into cytosol (von Eichel-Streiber et al., 1996). Vaccination is an effective means for the control of this disease. Whole-cell and toxoid vaccines are available, yet they have drawbacks such as incomplete inactivation and possible side effects. The use of recombinant DNA technology could result in antigens with high purity and safety (Fox and Klass, 1989). Protective antibodies against this recombinant antigen could react to the whole antigen and effective vaccines, producing the need to identify B-cell epitopes (Atassi et al., 2012). Therefore, epitope prediction is useful for the production of subunit and synthetic peptide vaccines and antibodies or antisera for competition assays. Identification of B-cell epitope locations has been carried out with both experimental and computational methods. In some experimental approaches, overlapping peptide was synthesized and bound to the antibody against the whole antigen (Atassi et al., 2011). Experimental techniques are time-consuming and expensive; furthermore, in the case of large proteins, it is laborious to carry out epitope mapping. Therefore, several prediction tools have been developed for identifying appropriate parts of protein as epitopes (Irving et al., 2001). This prediction is based on using computational methods and resources to extract immunological information and is known as immunoinformatics (Tomar and De, 2014). In this study, we designed a strategy to produce highly antigenic regions based on immunoinformatics methods in the recognized epitopic region, according to continuous, discontinuous, and T-cell epitopes. Production of antibodies was elicited by the immunization of mice

In Silico Vaccine Design: Targeting Highly Epitopic Regions ofClostridium perfringensType D Epsilon Toxin andClostridium novyiType B Alpha Toxin for Optimal Immunogenicity

“…The alignment of our selected fragment, ATXF (1822-1992 aa), with the highly antigenic C-terminal segment of ATX, specifically pNTF12 (1800-1958 aa), further supports the rationale behind our immunoinformatic design. According to research by Busch et al and Gord-Noshahri et al (Busch et al, 2000; Gord-Noshahri et al, 2016), pNTF12 is identified as a hydrophilic surface protein responsible for receptor binding activity. It exhibits high mobility and lacks catalytic activity.…”

“…Clostridium novyi is the causative organism for gangrene in humans and animals (Gord-Noshahri et al, 2016). Based on the production of different toxins, C. novyi is categorized into 4 types (A-D) (Oakley et al, 1947, 1959; Willis & others, 1964).…”

“…ATX, a 200–250 kDa protein (2178 aa residues) belongs to the large clostridial cytotoxin family (Hofmann et al, 1995) and acts as a glycosyltransferase on Rho proteins in target cells. It disintegrates the cytoskeleton through inhibiting signal pathways resulting in vascular permeability and cell death (Amimoto et al, 1998; Bette et al, 1991; Gord-Noshahri et al, 2016; Hofmann et al, 1995).…”

“…Furthermore, improper inactivation of toxoids can lead to side effects. Given that both bacteria are anaerobes, cultivation and preparation of toxins become laborious and expensive (Aquino et al, 2016; Chandran et al, 2010; Gord-Noshahri et al, 2016; Kaushik et al, 2019b; Petit et al, 2003).…”

Vaccination against pathogenic clostridia in animals: a review

“…Also, several experimental recombinant vaccines of C. novyi alpha toxin have been prepared and evaluated using immunological methods. Results have shown that antibody produced against recombinant proteins was more effective than that of normal toxin (Daryaei et al 2017 ; Noshahri et al 2016 ).…”